Tissue specific alternative splicing plays a prominent role in regulating gene expression in metazoans. Although the molecular events required to splice a simple pre-mRNA in vitro are fairly well understood, we remain largely ignorant of how the complex process of splicing is regulated in vivo. An understanding of how this process works is of fundamental importance since errors in RNA splicing are associated with a variety of human diseases, including cancer. The long-term goal of this project is to understand regulation by exon skipping, one of the most common forms of alternative splicing in humans. Specifically, we study the splicing regulation of the Drosophila developmental switch gene Sex-lethal (Sxl). Based on our earlier studies, we propose a novel mechanism by which the female specific product of the Sxl gene controls its own splicing pattern by interacting with, and inactivating, core components of the spliceosome. As outlined in the proposal, we are continuing to exploit Drosophila genetics to identify and delineate the function of proteins that participate in this biologically important example of splicing regulation. We are also using biochemical approaches to test the models that are known to be compatible with what we observe in vivo. Lastly, we are investigating, for the first time, whether the mechanism utilized to control Sxl is tissue-, as well as sex-specific. The combined data from these studies will provide insights into the complexities associated with splicing regulation that simply cannot be obtained via studies carried out solely in tissue culture cells. Due to the remarkable conservation of the splicing machinery from humans to flies, the paradigms we develop using the very tractable Drosophila system are relevant to human biology.